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The Biochemical Journal Jul 1992Low 2H2O effects (1.0-1.5) for the parameter k(cat.)/Km in the hydrolysis of various substrates by acetylcholinesterase (AcChE) is due to normal 2H2O effects (1.8-2.8)...
Low 2H2O effects (1.0-1.5) for the parameter k(cat.)/Km in the hydrolysis of various substrates by acetylcholinesterase (AcChE) is due to normal 2H2O effects (1.8-2.8) for the parameter k(cat.) and 2H2O effects of 1.0-2.5 for the parameter Km. The analyses and interpretations of 2H2O effects in the literature utilizing the parameter k(cat.)/Km, which led to the proposal of 'isotope insensitivity' of the catalytic steps and the hypothesis of a rate-limiting substrate-induced-fit conformational change, are incorrect. Since k(cat.) is the only parameter that can represent the hydron-transfer step solely, the 2H2O effect can most appropriately be evaluated by using this parameter. Calculations and comparison of acylation (k+2) and deacylation (k+3) rate constants show that acylation is rate-determining for most substrates and the improved binding -0.84 to -2.09 kJ/mol (-0.2 to -0.5 kcal/mol) in 2H2O obscures the normal 2H2O effect on k(cat.) when the ratio k(cat.)/Km is utilized. Consistent with this, measurements of the inhibition constant (KI(com.)) for a reversible inhibitor, phenyltrimethylammonium, lead to KI(com.)H2O = 39 +/- 3 microM and KI(com.)2H2O = 24.5 +/- 3.5 microM, an 2H2O effect of 1.59 +/- 0.26. pH-dependence of k(cat.) in 2H2O is subject to variability of the pK(app.) values, as evaluated in terms of the two-hydronic-reactive states (EH and EH2) of AcChE, and is due to an uneven decrease in 2H2O of the kinetic parameters k'cat. for the EH2 state relative to k(cat.) for the EH state, thus leading to variable shifts in pK(app.) values of between 0.5 and 1.2 pH units for this parameter. The observed pH-independent limiting rate constants for k(cat.)/Km(app.) are made to vary between 0.5 and 1.0 in 2H2O by effects on kinetic parameters for the EH2 state, k'cat./K'm varying between 0.2 and 0.7 relative to the EH state, with k(cat.)/Km varying between 0.4 and 1.0. The effects observed on k(cat.)/Km(app.) are ultimately the result of variable effects of 2H2O on k'cat. and K'm for the EH2 state relative to k(cat.) and Km for the EH state of AcChE. These effects are responsible for the variable shifts and more than 0.5 pH unit of the pK(app.) values in 2H2O for pH-k(cat.)/Km profiles. The upward-bowing hydron inventories for k(cat.)/Km are the result of linear hydron inventories for k(cat.) and downward-bowing on Km and are not due to the rate-limiting substrate-induced fit process as claimed in the literature.(ABSTRACT TRUNCATED AT 400 WORDS)
Topics: Acetylcholinesterase; Acylation; Catalysis; Deuterium; Deuterium Oxide; Hydrogen-Ion Concentration; Kinetics; Substrate Specificity; Water
PubMed: 1322133
DOI: 10.1042/bj2850451 -
Langmuir : the ACS Journal of Surfaces... Sep 2016Ice nucleation is of fundamental significance in many areas, including atmospheric science, food technology, and cryobiology. In this study, we investigated the...
Ice nucleation is of fundamental significance in many areas, including atmospheric science, food technology, and cryobiology. In this study, we investigated the ice-nucleation characteristics of picoliter-sized drops consisting of different D2O and H2O mixtures with and without the ice-nucleating bacteria Pseudomonas syringae. We also studied the effects of commonly used cryoprotectants such as ethylene glycol, propylene glycol, and trehalose on the nucleation characteristics of D2O and H2O mixtures. The results show that the median freezing temperature of the suspension containing 1 mg/mL of a lyophilized preparation of P. syringae is as high as -4.6 °C for 100% D2O, compared to -8.9 °C for 100% H2O. As the D2O concentration increases every 25% (v/v), the profile of the ice-nucleation kinetics of D2O + H2O mixtures containing 1 mg/mL Snomax shifts by about 1 °C, suggesting an ideal mixing behavior of D2O and H2O. Furthermore, all of the cryoprotectants investigated in this study are found to depress the freezing phenomenon. Both the homogeneous and heterogeneous freezing temperatures of these aqueous solutions depend on the water activity and are independent of the nature of the solute. These findings enrich our fundamental knowledge of D2O-related ice nucleation and suggest that the combination of D2O and ice-nucleating agents could be a potential self-ice-nucleating formulation. The implications of self-nucleation include a higher, precisely controlled ice seeding temperature for slow freezing that would significantly improve the viability of many ice-assisted cryopreservation protocols.
Topics: Cryoprotective Agents; Deuterium Oxide; Emulsions; Ice; Oils; Pseudomonas syringae
PubMed: 27495973
DOI: 10.1021/acs.langmuir.6b02212 -
European Journal of Applied Physiology Jun 2012The kinetic parameters of absorption and distribution of ingested water (300 ml labeled with D(2)O; osmolality <20 mOsm kg(-1)) in the body water pool (BWP) and of its... (Randomized Controlled Trial)
Randomized Controlled Trial
The kinetic parameters of absorption and distribution of ingested water (300 ml labeled with D(2)O; osmolality <20 mOsm kg(-1)) in the body water pool (BWP) and of its disappearance from this pool were estimated in 36 subjects from changes in plasma or urine deuterium to protium ratio (D/H) over 10 days using one- and two-compartment and a non-compartmental pharmacokinetic models (1-CM, 2-CM and N-CM which applied well to 58, 42 and 100% of the subjects, respectively). Compared with the volume and turnover of the BWP computed with the slope-intercept method (60.7 ± 4.1% body mass or 72.7 ± 3.2% lean body mass; turnover 4.58 ± 0.80 l day(-1): i.e., complete renewal in ~50 days; n = 36), the values were accurately estimated with the N-CM and 1-CM and were slightly overestimated and underestimated, respectively, with the 2-CM (~7-8% difference, significant for water clearance only). Ingested water appeared in plasma and blood cells within 5 min and the half-life of absorption (~11-13 min) indicates a complete absorption within ~75-120 min. The 2-CM showed that in 42% of the subjects, ingested water quickly distributed within a central compartment before diffusing with a very short half-life (12.5 ± 4.3 min) to a peripheral compartment (18.5 ± 4.3 and 31.6 ± 6.4 L, respectively), which were in complete equilibrium within ~90 min. Pharmacokinetic analyses of water labeled with D(2)O can help describe water absorption and distribution, for which there is no well defined reference method and value; depending on the characteristics of the subjects and the drinks, and of environmental conditions.
Topics: Absorption; Adult; Body Fluids; Deuterium Oxide; Humans; Kinetics; Male; Tissue Distribution; Water
PubMed: 21997675
DOI: 10.1007/s00421-011-2194-7 -
Journal of Dairy Science Apr 2017Deuterium oxide (DO) dilution methods have been used to assess body composition in live animals. Estimated body water content can be used to predict body fat and... (Randomized Controlled Trial)
Randomized Controlled Trial
Deuterium oxide (DO) dilution methods have been used to assess body composition in live animals. Estimated body water content can be used to predict body fat and protein, and thus, the amount of energy reserves. It is an alternative method to direct chemical analysis and considered a noninvasive technique that is economical and repeatable. Deuterium oxide use is considered easy, safe, and accurate; however, the traditional methods of analyzing DO are expensive, tedious, and time consuming. The objective of this study was to evaluate the potential for using nuclear magnetic resonance spectroscopy (NMR) to determine body composition in Holstein dairy heifers. Nuclear magnetic resonance is less expensive and requires minutes to calculate the percentage of DO in the blood. This study used 24 newborn dairy heifer calves blocked by birth and randomly assigned to 1 of 3 treatments: (1) 446 g dry matter (DM) of a conventional milk replacer (MR) [CON; 20% crude protein (CP), 20% fat], (2) 669 g DM of a moderately high protein MR (MOD; 26% CP, 18% fat), or (3) 892 g DM of a moderately high protein MR (aggressive, AGG; 26% CP, 18% fat). All calves had free-choice access to starter and water. Both MR and starter were medicated with decoquinate. During weaning (d 43 to 49), the morning MR feeding ceased. On d 50, all MR feedings ended but starter and water intakes were continuously recorded until d 56. When calves were 50 d of age, a baseline blood sample was taken followed by injection of 300 mg of DO/kg of body weight in sterile physiological saline (0.9%). The syringes containing the DO in physiological saline were weighed before and after administration to record the actual dose of DO injected gravimetrically. After injection, the DO was allowed to equilibrate with body water for 1 h. Six blood samples were taken over 6 d (1/d) at 1630 h to estimate the dilution of the tracer. The plasma was aspirated and stored at -20°C until further DO analysis. This new method was validated using 4 calf plasma samples that were sent to an outside laboratory for measurement using an independent validation method. We detected no differences in total body water, protein, fat, or mineral content in calves fed CON, MOD, or AGG; however, results demonstrated that the DO dilution technique and analysis by NMR is an appropriate and easy method to estimate water, protein, ash, and fat in young heifers.
Topics: Animal Feed; Animals; Body Composition; Cattle; Deuterium Oxide; Diet; Female; Magnetic Resonance Spectroscopy; Weaning
PubMed: 28161168
DOI: 10.3168/jds.2016-11888 -
FEMS Microbiology Ecology Oct 2015The combined approach of incubating environmental samples with stable isotope-labeled substrates followed by single-cell analyses through high-resolution secondary ion...
The combined approach of incubating environmental samples with stable isotope-labeled substrates followed by single-cell analyses through high-resolution secondary ion mass spectrometry (NanoSIMS) or Raman microspectroscopy provides insights into the in situ function of microorganisms. This approach has found limited application in soils presumably due to the dispersal of microbial cells in a large background of particles. We developed a pipeline for the efficient preparation of cell extracts from soils for subsequent single-cell methods by combining cell detachment with separation of cells and soil particles followed by cell concentration. The procedure was evaluated by examining its influence on cell recoveries and microbial community composition across two soils. This approach generated a cell fraction with considerably reduced soil particle load and of sufficient small size to allow single-cell analysis by NanoSIMS, as shown when detecting active N2-fixing and cellulose-responsive microorganisms via (15)N2 and (13)C-UL-cellulose incubations, respectively. The same procedure was also applicable for Raman microspectroscopic analyses of soil microorganisms, assessed via microcosm incubations with a (13)C-labeled carbon source and deuterium oxide (D2O, a general activity marker). The described sample preparation procedure enables single-cell analysis of soil microorganisms using NanoSIMS and Raman microspectroscopy, but should also facilitate single-cell sorting and sequencing.
Topics: Archaea; Bacteria; Carbon; Deuterium Oxide; Fungi; Isotope Labeling; RNA, Ribosomal, 16S; Single-Cell Analysis; Soil Microbiology; Spectrometry, Mass, Secondary Ion; Spectrum Analysis, Raman
PubMed: 26324854
DOI: 10.1093/femsec/fiv106 -
The Journal of Clinical Investigation Feb 1954
Topics: Body Fluids; Body Water; Deuterium Oxide; Female; Pregnancy; Research; Water
PubMed: 13130691
DOI: 10.1172/JCI102890 -
Canadian Journal of Chemistry Feb 1951
Topics: Deuterium; Deuterium Oxide; Hydrogen Peroxide; Inorganic Chemicals; Organic Chemicals; Peroxides; Refractometry; Solutions; Surface Tension; Viscosity
PubMed: 14821849
DOI: 10.1139/v51-022 -
Nature Communications Mar 2020Enzymes dependent on nicotinamide cofactors are important components of the expanding range of asymmetric synthetic techniques. New challenges in asymmetric catalysis...
Enzymes dependent on nicotinamide cofactors are important components of the expanding range of asymmetric synthetic techniques. New challenges in asymmetric catalysis are arising in the field of deuterium labelling, where compounds bearing deuterium (H) atoms at chiral centres are becoming increasingly desirable targets for pharmaceutical and analytical chemists. However, utilisation of NADH-dependent enzymes for H-labelling is not straightforward, owing to difficulties in supplying a suitably isotopically-labelled cofactor ([4-H]-NADH). Here we report on a strategy that combines a clean reductant (H) with a cheap source of H-atoms (HO) to generate and recycle [4-H]-NADH. By coupling [4-H]-NADH-recycling to an array of C=O, C=N, and C=C bond reductases, we demonstrate asymmetric deuteration across a range of organic molecules under ambient conditions with near-perfect chemo-, stereo- and isotopic selectivity. We demonstrate the synthetic utility of the system by applying it in the isolation of the heavy drug (1S,3'R)-[2',2',3'-H]-solifenacin fumarate on a preparative scale.
Topics: Biocatalysis; Chemistry Techniques, Synthetic; Deuterium; Deuterium Oxide; Isotope Labeling; Molecular Structure; Niacinamide; Oxidoreductases; Solifenacin Succinate; Stereoisomerism
PubMed: 32193396
DOI: 10.1038/s41467-020-15310-z -
The Production of Matchout-Deuterated Cholesterol and the Study of Bilayer-Cholesterol Interactions.Scientific Reports Mar 2019The deuteration of biomolecules provides advanced opportunities for neutron scattering studies. For low resolution studies using techniques such as small-angle neutron...
The deuteration of biomolecules provides advanced opportunities for neutron scattering studies. For low resolution studies using techniques such as small-angle neutron scattering and neutron reflection, the level of deuteration of a sample can be varied to match the scattering length density of a specific DO/HO solvent mixture. This can be of major value in structural studies where specific regions of a complex system can be highlighted, and others rendered invisible. This is especially useful in analyses of the structure and dynamics of membrane components. In mammalian membranes, the presence of cholesterol is crucial in modulating the properties of lipids and in their interaction with proteins. Here, a protocol is described for the production of partially deuterated cholesterol which has a neutron scattering length density that matches that of 100% DO solvent (hereby named matchout cholesterol). The level of deuteration was determined by mass spectrometry and nuclear magnetic resonance. The cholesterol match-point was verified experimentally using small angle neutron scattering. The matchout cholesterol was used to investigate the incorporation of cholesterol in various phosphatidylcholine supported lipid bilayers by neutron reflectometry. The study included both saturated and unsaturated lipids, as well as lipids with varying chain lengths. It was found that cholesterol is distributed asymmetrically within the bilayer, positioned closer to the headgroups of the lipids than to the middle of the tail core, regardless of the phosphatidylcholine species.
Topics: Cholesterol; Deuterium Oxide; Lipid Bilayers; Neutron Diffraction; Scattering, Small Angle
PubMed: 30914734
DOI: 10.1038/s41598-019-41439-z -
Molecules (Basel, Switzerland) Dec 2018Deuterium oxide (D₂O) has been reported to be active toward various in vitro cell lines in combination with phytochemicals. Our objective was to describe, for the...
Deuterium oxide (D₂O) has been reported to be active toward various in vitro cell lines in combination with phytochemicals. Our objective was to describe, for the first time, the effect of D₂O on the proliferation of hepatic stellate cells (HSCs). After D₂O treatment, the p53-cyclin-dependent kinase (CDK) pathway was stimulated, leading to inhibition of the proliferation of HSCs and an increase in the [ATP]/[ADP] ratio. We also evaluated the role of aquaporin (AQP) 11 in activated HSCs. We found that D₂O treatment decreased AQP11 expression levels. Of note, AQP11 levels elevated by a genetic approach counteracted the D₂O-mediated inhibition of proliferation. In addition, the expression levels of AQP11 negatively correlated with those of p53. On the other hand, cells transfected with an AQP11-targeted small interfering RNA (siRNA) showed enhanced inhibition of proliferation. These findings suggest that the inhibition of cell proliferation by D₂O in activated HSCs could be AQP11 dependent. Our previous studies have documented that bisdemethoxycurcumin (BDMC) induces apoptosis by regulating heme oxygenase (HO)-1 protein expression in activated HSCs. In the current study, we tested whether cotreatment with BDMC and D₂O can modulate the AQP11-dependent inhibition of cell proliferation effectively. We observed that D₂O cotreatment with BDMC significantly decreased cell proliferation compared to treatment with D₂O alone, and this effect was accompanied by downregulation of HO-1 and an increase in p53 levels.
Topics: Adenosine Triphosphate; Animals; Apoptosis; Aquaporins; Cell Cycle Checkpoints; Cell Line; Cell Line, Tumor; Cell Proliferation; Cells, Cultured; Cyclins; Deuterium Oxide; Heme Oxygenase-1; Hepatic Stellate Cells; Humans; Rats; Signal Transduction; Tumor Suppressor Protein p53
PubMed: 30563120
DOI: 10.3390/molecules23123209